• Anaerobic Toxicity Assay
    for AD/RNG Feedstocks
    At Celignis Biomass Lab

The Importance of Identifying Toxic Feedstocks for AD and RNG

Anaerobic Digestion (AD) can be a very effective technology for valorising a wide variety of feedstocks and waste streams, allowing for revenue generation from materials and side-streams that may otherwise require costly waste-disposal procedures. However, it is important to recognise that some feedstocks may not be suitable at certain concentrations and may give rise to performance issues in the digester, leading to lower biogas and biomethane yields and, potentially, to the failure of the renewable natural gas (RNG) system.

This is because the microbiome in the digester is complex and consists of a variety of bacteria and archaea systems. Any alteration in this microbiome can lead to inefficient progress through the stages of hydrolysis, acidogenesis, acetogenesis, and methanogenisis. This will lead to accumulation of inhibitory metabolites in the digester.

Some examples of scenarios where certain feedstocks can be toxic to the digester are provided below:

Cattle slurry from some sources can have high contents of antibiotics and their presence in the digester can lead to its underperformance or even to complete failure is such slurry is added beyond a certain threshold proportion of the feedstock mix.

DAF (Dissolved Air Flotation) sludge, arising from aerobic treatment systems, can be used a feedstock for AD. However, the polyelectrolytes that are used in the DAF process can contain high contents of aluminium that can lead to aluminium toxicity issues within the digester.

Industry wash streams, used to clean process reactors, can contain detergents and chlorine-containing agents. These wash streams form a certain percentage of the industry stream sent for AD. When such a stream is to be treated by anaerobic digestion the residual chlorine and detergents cause toxicity to the microbes in the reactor which can lead to digester failure.

Anaerobic Toxicity Assay (ATA)

At Celignis we have designed a custom assay to evaluate whether selected feedstocks can potentially be toxic for anaerobic digestion. This ATA (anaerobic toxicity assay) test has been developed based on our experience with analysing and processing a wide variety of waste streams.

The assay involves the use of standard media containing glucose as the primary media to which the test stream is added in different concentrations. The negative or positive effect assaciated with the addition of the test stream is calculated based on the amount of biogas produced per gram glucose in the presence or absence of the test stream.

The ATA runs for 5 days, using the same equipment we use to undertake our biomethane potential analysis of biogas feedstocks. Our reports summarise the differences seen between the different feedstock loadings and also include graphs compariong the daily and cumulative biogas production, as illustrated here.

The anaerobic toxicity assay not only indicates whether a feedstock can potentially have toxic effects on the anaerobic digestion process but, since we perform the test at different feedstock concentrations, it also allows us to suggest at which concentrations the feedstock can be safely added. This will help you to effectively proportion the feedstock mixes in your renewable natural gas process.

Additionally, if inhibition is detected then we can, if requested, work further on looking to identify the source of this inhibition. Such analysis may include undertaking a minerals analysis or looking at the anions and metals present. Also, in the case of streams where antibiotics may be present, we can perform microbial-disc assays to identify the zone of inhibition, allowing us understand the extent of inhibition in the system.

Celignis ATA Packages

The Celignis Analysis Package(s) that include the Anaerobic Toxicity Assay are listed below:

Celignis Case Study Involving Anaerobic Toxicity Assays

A biogas plant started underperforming when a new feedstock was used as co-feed to the plant. As the plant received an important gate-fee for this new feedstock, they did not want to discontinue its use but to instead use it in a controlled and scientifically-driven manner. Celignis was asked to provide support for: determining the toxic effects of the feedstock; the causes of it; and to provide feeding limits.

Celignis undertook chemical analyses on the feedstock and then custom-designed and performed Anaerobic Toxicity Assays for the waste stream based on the analytical data. These experiments allowed for the determination of threshold feedstock-loadings, as co-feed, in order to avoid the toxic/inhibitory effects of the feedstocks.

The biogas plant is now benefiting from Celignis's support since they can make informed decisions on using feedstocks coming various process industries and so can maintain healthy digestion while incorporating new waste streams into the feed-mix of the RNG plant.

Click here for information on our Process Optimisation services.

Additional Information on Anaerobic Toxicity Assay

Feel free to get in touch with us if you have any questions about our Anaerobic Toxicity Assay or if you think that some of the feedstocks that you are considering for biogas production and Renewable Natural Gas (RNG) projects may be potentially toxic. Relevant members of the Celignis anaerobic digestion team will be happy to assist. Those team members with the most experience with undertaking this test and interpreting the resulting data are listed below.

Lalitha Gottumukkala

Founder and Lead of Celignis AD, CIO of Celignis


Has a deep understanding of all biological and chemical aspects of anaerobic digestion. Has developed Celignis into a renowned provider of AD services to a global network of clients.

Kwame Donkor

AD Services Manager

BSc, MSc, Phd (yr 4)

His PhD focused on optimising AD conditions for Irish feedstocks such as grass. Kwame is now leading the Celignis AD team in the provision of analysis and bioprocess services.

Piotr Dobkowski

Orders and Data Manager


Feeds on quality data! Piotr plays a major role in data processing and Orders management at Celignis and is responsible for ensuring AD data are rapidly uploaded to the Celignis Database.

Other Celignis Tests and Services for Anaerobic Digestion

Global Recognition as AD/RNG Experts

Celignis provides valued services to over 1000 clients. We understand how the focus of AD projects can differ between countries and have advised a global network of clients on their RNG projects. We also have customs-exemptions for samples sent to us allowing us to quickly get to work no matter where our clients are based.

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Biomethane Potential

The biomethane potential (BMP) can be considered to be the experimental theoretical maximum amount of methane produced from a feedstock. In our laboratory, we have six BMP systems, comprising 90 reactors, that allow us to digest your samples and determine the biogas yield over periods of between 14 and 40 days.

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Continuous Digestions

To help you evaluate how well your anaerobic digestion feedstocks will behave in real-world conditions we can undertake continuous digestion experiments. These operate at scales up to 12 litres and typically run for 3 months. We target maximum achievable organic loading rate (OLR) and biomethane potential.

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Process Optimisations

There a many factors to consider when running an AD facility. We can design and experimentally-validate optimisations of these factors at the lab-scale prior to you implementing them at your AD facility. Such an approach allows for greater benefits and lower costs than optimising the process at the commercial scale.

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Feedstock Analysis

Our analysts have characterised tens of thousands of biomass samples. We have dedicated analyses packages for the compositional parameters of most relevance to AD/RNG. Additionally, based on our detailed analyses we can recommend appropriate feedstock mixing proportions in co-digestion facilities.

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Biological Consultations

We're experts in the biology of anaerobic digestion. We pour through operational data from biogas plants and identify correlations between process parameters and plant performance. This understanding on the specific biology of the digester allows for recommendations as to how peformance can be improved and made more stable.

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Specific Microbial Activity

AD is a microbial process involving a sequence of stages (hydrolysis, acidogenesis, methanogenesis) to convert a complex feedstock to methane. We analyse samples collected from digesters and undertake tests to investigate how well they proceed with each of these stages of digestion.

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Technoeconomic Analyses

Our TEA experts work with you to evaluate the economic prospects of your AD/RNG facility, considering various scale, technology, and feedstock options. We apply accurate costing models to determine CAPEX/OPEX of simulated and pilot scale processes which are then used to determine key economic indicators (e.g. IRR, NPV).

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Digestate Analysis

Digestate is the residue after the anaerobic digestion process. It can potentially have value as a soil fertiliser. We offer a range of detailed analysis packages for digestate, allowing you to fully assess this resource and to determine the best use for it. Our team can also assist in evaluating digestate valorisation options.

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Project Development

The criteria for the development of a successful AD project are numerous and vary according to region, technology, and feedstock. We have a deep understanding of these regional, technical, and biological differences and have advised a global network of clients on effectively developing their AD projects.

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Process Parameters

Celignis can undertake a range of key analyses for KPIs and advanced process monitoring. These include volatile fatty acids (VFAs); Alkalinity ratio (FOS/TAC); and redox potential. It is particularly imporant that these are monitored when undergoing changes of feedstock type, organic loading rate and hydraulic retention times.

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Nutrient Supplementations

Nutrients are essential for maintaining stable microbial populations and for efficient anaerobic digestion. We can suggest optimal values for the presence of major and minor elements in the digester as well as upper and lower threshold values. This allows us to formulate a bespoke cocktail of additives according to the requirements of the digester.

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Publications on Anaerobic Digestion By The Celignis Team

Ravindran, R., Donkor, K., Gottumukkala, L., Menon, A., Guneratnam, A. J., McMahon, H., Koopmans, S., Sanders, J. P. M., Gaffey, J. (2022) Biogas, biomethane and digestate potential of by-products from green biorefinery systems, Clean Technologies 4(1): 35-50



Global warming and climate change are imminent threats to the future of humankind. A shift from the current reliance on fossil fuels to renewable energy is key to mitigating the impacts of climate change. Biological raw materials and residues can play a key role in this transition through technologies such as anaerobic digestion. However, biological raw materials must also meet other existing food, feed and material needs. Green biorefinery is an innovative concept in which green biomass, such as grass, is processed to obtain a variety of protein products, value-added co-products and renewable energy, helping to meet many needs from a single source. In this study, an analysis has been conducted to understand the renewable energy potential of green biorefinery by-products and residues, including grass whey, de-FOS whey and press cake. Using anaerobic digestion, the biogas and biomethane potential of these samples have been analyzed. An analysis of the fertiliser potential of the resulting digestate by-products has also been undertaken. All the feedstocks tested were found to be suitable for biogas production with grass whey, the most suitable candidate with a biogas and biomethane production yield of 895.8 and 544.6 L/kg VS, respectively, followed by de-FOS whey and press cake (597.4/520.3 L/kg VS and 510.7/300.3 L/kg VS, respectively). The results show considerable potential for utilizing biorefinery by-products as a source for renewable energy production, even after several value-added products have been co-produced.

Donkor, K. O., Gottumukkala, L. D., Lin, R., Murphy, J. D. (2022) A perspective on the combination of alkali pre-treatment with bioaugmentation to improve biogas production from lignocellulose biomass, Bioresource Technology 351



Anaerobic digestion (AD) is a bioprocess technology that integrates into circular economy systems, which produce renewable energy and biofertilizer whilst reducing greenhouse gas emissions. However, improvements in biogas production efficiency are needed in dealing with lignocellulosic biomass. The state-of-the-art of AD technology is discussed, with emphasis on feedstock digestibility and operational difficulty. Solutions to these challenges including for pre-treatment and bioaugmentation are reviewed. This article proposes an innovative integrated system combining alkali pre-treatment, temperature-phased AD and bioaugmentation techniques. The integrated system as modelled has a targeted potential to achieve a biodegradability index of 90% while increasing methane production by 47% compared to conventional AD. The methane productivity may also be improved by a target reduction in retention time from 30 to 20 days. This, if realized has the potential to lower energy production cost and the levelized cost of abatement to facilitate an increased resource of sustainable commercially viable biomethane.

Donkor, K. O., Gottumukkala, L. D., Diedericks, D., Gorgens, J. F. (2021) An advanced approach towards sustainable paper industries through simultaneous recovery of energy and trapped water from paper sludge, Journal of Environmental Chemical Engineering 9(4): 105471


This study considered the possibility of reducing the environmental footprint of paper and pulp industry by producing bioenergy from paper sludge by using process wastewater instead of fresh water, and reclaiming water trapped in paper sludge. Experimental studies are conducted with streams from three different pulp and paper mills (virgin pulp mill (VP), corrugated recycling mill (CR), tissue printed recycling mill (TPR)) for sequential bioethanol and biogas production with simultaneous reclamation of water from paper sludge (PS). Total energy yields of 9215, 6387, 5278 MJ/tonne dry PS for VP, CR and TPR, respectively, were obtained for ethanol-biogas production. Virgin pulp paper sludge gave the highest yield for ethanol and biogas in stand-alone processes (275.4 kg and 67.7 kg per ton dry PS respectively) and also highest energy conversion efficiency (55%) in sequential process compared with CR and TPR. Energy and environmental case study conducted on virgin pulp mill has proven the possibility of using paper sludge bioenergy to reduce energy demand by 10%, while reclaiming 82% of the water from the PS, reducing greenhouse gas emissions (GHG) by 3 times and producing solids suitable for land spreading.

Gottumukka L.D, Haigh K, Collard F.X, Van Rensburg E, Gorgens J (2016) Opportunities and prospects of biorefinery-based valorisation of pulp and paper sludge, Bioresource technology 215: 37-49


The paper and pulp industry is one of the major industries that generate large amount of solid waste with high moisture content. Numerous opportunities exist for valorisation of waste paper sludge, although this review focuses on primary sludge with high cellulose content. The most mature options for paper sludge valorisation are fermentation, anaerobic digestion and pyrolysis. In this review, biochemical and thermal processes are considered individually and also as integrated biorefinery. The objective of integrated biorefinery is to reduce or avoid paper sludge disposal by landfilling, water reclamation and value addition. Assessment of selected processes for biorefinery varies from a detailed analysis of a single process to high level optimisation and integration of the processes, which allow the initial assessment and comparison of technologies. This data can be used to provide key stakeholders with a roadmap of technologies that can generate economic benefits, and reduce carbon wastage and pollution load.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Pandey A (2014) Growth and butanol production by Clostridium sporogenes BE01 in rice straw hydrolysate: kinetics of inhibition by organic acids and the strategies for their removal, Biomass Conversion and Biorefinery 4(3): 277-283


Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resins—XAD 4, XAD 7, XAD 16, and an anion exchange resin—Seralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.